Project Summary/Abstract Recent work by our laboratory has identified a delimited node of VGAT+ (GABAergic) neurons in the ventral lateral hypothalamus (LHVGAT) that are both necessary and sufficient for normal behavioral and electroen- cephalographic (EEG) wakefulness. There is a fundamental gap however in understanding the cellular and synaptic circuit basis by which LHVGAT neurons trigger waking. The long-term goal is to understand the func- tional circuit basis by which LHVGAT neurons generate and regulate behavioral and EEG arousal. The objective in this particular application is to extend our previous findings by defining the functional, synaptic ?neurocircuit? basis by which LHVGAT neurons trigger wakefulness. The central hypothesis is that LHVGAT neurons promote wake and fast EEG rhythms through direct inhibition of the sleep-active ventrolateral preoptic (VLPO) nucleus, located within the greater preoptic area. The rationale for the proposed research is that identifying the circuit basis by which LHVGAT neurons promote waking represents a critical first step towards manipulating them and reducing the dysfunction experienced by individuals with arousal-based disorders, including hyperarousal- driven insomnia. Guided by strong preliminary data, our hypotheses will be tested by pursuing three specific aims: 1) determine if LHVGAT neurons promote arousal through direct inhibition of sleep-promoting VLPO neu- rons; 2) define the functional synaptic physiology of the LHVGAT?VLPO interface, including the cellular profile of the targeted neurons; and 3) determine synaptic inputs to the LHVGAT and establish a functional tri-synaptic cir- cuit spanning input?LHVGAT?VLPO. The approach is intellectually and technically innovative because it rep- resents a new and substantive departure from contemporary models of the role of LH neurons in wake-sleep regulation and because it employs a novel combination of newly developed and validated approaches, includ- ing complimentary in vivo and in vitro opto-genetic based experiments. The proposed research is significant because it is expected to vertically advance and expand understanding of the cellular and circuit (synaptic) mechanisms underlying LHVGAT regulation of wakefulness and fast cortical rhythms associated with cognition. Ultimately, such knowledge has the potential to inform the development of therapeutic and interventional strat- egies to reduce the dysfunction and negative health effects experienced by a growing number of patients worldwide with arousal-based disorders and disorders of arousal and sleep, including hyperarousals of PTSD and insomnia.